Peroxidation of polyunsaturated fatty acids (PUFAs) and their biologically relevant phospholipid esters is a complex reaction giving scores of possible products from a single molecular species. This process is a hallmark of diverse environmental chemical exposures, drug toxicities and oxidative stresses. In addition to the many peroxide products that form from polyunsaturated lipids, a set of reactive electrophiles is also generated. These electrophilic residuals of lipid peroxidation modify nucleic acids and proteins and in this way, the consequence of lipid oxidative degradation is distributed to other important biomolecules. This proposal outlines experiments that probe the chemical mechanisms of lipid peroxidation, provides a framework for understanding the oxidation of highly unsaturated lipids present in fish oils, describes novel new affinity-tags useful in the isolation of lipid electrophile-protein adducts and examines new phenolic antioxidants more potent than vitamin E. Highly unsaturated co-3 PUFAs are better reducing agents than more saturated co-6 lipids and we will look for consequences of this difference by analyzing peroxidation biomarkers formed in model membrane oxidations and in tissues and fluids of stressed animals on fish oil diets. Our affinity-tag lipids are analogs of natural lipids having a terminal alkyne substituted at the 00 position (co-yne) of fatty acid chains. This terminal alkyne undergoes click cycloaddition with biotinsubstituted azides, permitting pull-down of any proteins covalently attached to lipid-derived electrophiles bearing an (co-yne). The proposed research is based on the hypothesis that the chemical mechanisms of lipid peroxidation and the formation of electrophilic byproducts that are a hallmark of this process can be rationally defined. The affinity tags when coupled to powerful HPLC/MS/MS proteomics methods permit the structural identification of individual lipid-protein adducts even though such species are only a small part of a very complex mixture. Profiling of human THP-1 cells exposed to an oxidative stress will include studies in which affinity tag (co-yne) lipids are incorporated into the cells, permitting isolation of lipid-protein adducts. The electrophiles identified from phospholipids will form the basis of a screening program in collaboration with Projects 3 and 4 of the Program Project. New powerful pyridinol antioxidants will be studied in proof of concept in vivo rodent experiments.